Genomic amplification strategies using the polymerase chain reaction (PCR; Mullis & Faloona, 1987, Meth. Enzymol. 155:335) are employed to facilitate the identification of polymorphic sequences. PCR is used to amplify regions of genomic DNA that carry potential polymorphisms. One method hybridizes the PCR products to allele-specific hybridization probes (Saiki et al., 1986, Nature 324:163). Other methods utilize oligonucleotide primers that either match or mismatch the targeted polymorphism (Newton et al., 1989, Nucleic Acids Res. 17:2503).
With methods that hybridize the PCR product to an allele-specific probe, PCR is used to reduce the complexity of the DNA sample being assayed for the polymorphic marker and to increase the number of copies of the polymorphism-bearing DNA. If 100,000 polymorphic markers were to be assayed per genome, it would be very expensive to perform 100,000 individual PCR reactions. Some advances have been made to multiplex PCR reactions (Chamberlain et al., 1988, Nucl. Acids Res. 16:11141), and the degree of multiplexing of the PCR has been scaled up, followed by hybridization to an array of allele-specific probes (Wang et al., 1998, Science 280: 1077). However, in the studies by Wang et al., the percentage of PCR products that successfully amplified decreased as the number of PCR primers added to the reaction increased. When approximately 100 primer pairs were used, about 90% of the PCR products were successfully amplified. When the number of primer pairs was increased to about 500, about 50% of the PCR products were successfully amplified. Another disadvantage with multiplex PCR is that individual primer pairs must be synthesized for each polymorphic target. Genotyping DNA with 100,000 polymorphism targets would require, in theory, 200,000 different PCR primers. Not only is the synthesis of such primers costly and time consuming, but not all primer designs succeed in producing a desired PCR product. Therefore considerable time and energy may be spent optimizing the primer designs.
Hatada et al. have cleaved genomic DNA with a rarely cutting restriction enzyme, separated the cleaved DNA by gel electrophoresis, again cleaved the separated DNA with a second restriction enzyme in the gel, and again separated the DNA in a second dimension by electrophoresis (Hatada et al., 1991, Proc. Natl. Acad. Sci. USA 88: 9523). According to the Hatada et al. method, one then examines the two-dimensional pattern of DNA spots using DNA from different individuals. Differences in DNA migration patterns result from sequence or nucleotide methylation differences in the restriction enzyme recognition sequences.
Hayashizaki et al. (Hayashizaki et al., 1992, Genomics 14:733) use solid-phase adapters specific for restriction fragment ends to physically separate a subset of fragments from genomic DNA. After purification of the adapter-bound DNA fraction away from the rest of the genomic DNA, the bound DNA is separated from the adapters by cleaving again with the restriction enzyme used for the adapter ligation. The DNA released from the adapters is then cloned into a replication vector to make a gene library.
Others have used DNA binding factors to reduce the complexity of populations of synthetic oligonucleotides with stretches of randomized sequences, with the aim of elucidating the consensus binding sequences of the proteins (Mavrothalassitis et al., 1990, DNA Cell Biol., 9:783; Blackwell & Weintraub, 1990, Science, 250: 1104; Woodring et al., 1993, Trends Biol. Sci., 18: 77; Hardenbol & Van Dyke, 1996, Proc. Natl. Acad. Sci. U.S.A., 93: 2811).
There is a need in the art for improved methods of identifying polymorphic sequences.